Nucleic acid can be quantified using Polymerase Chain Reaction (PCR) by the detection of amplification products present at the end of PCR, endpoint quantitative PCR, or during PCR, quantitative real-time PCR (qrtPCR). In qrtPCR, fluorescent dyes are generally used to detect PCR products during thermal cycling. This allows quantification of the template to be based on the intensity of the fluorescent signal during the exponential phase of amplification; before limiting reagents or the inactivation of the polymerase have started to have an effect on the efficiency of PCR amplification.
Specialized instruments are used for qrtPCR assays. These qrtPCR instruments couple the thermal cycling function of PCR machines with a fluorimeter. This combination of a thermal cycling function and a fluorimeter allows for in-tube, real-time analysis.
qrtPCR is conducted by placing a reaction tube(s) in a qrtPCR instrument and subjecting the reaction tube(s) to thermal cycling. During each thermal cycling round, the reaction tube is illuminated with light and fluorescence emanating from a reaction tube is collected by the fluorimeter. Because the accuracy of qrtPCR depends on the measurement of fluorescent emissions factors that interfere or interrupt the optical pathway are minimized.
The dominant thinking has been that opaque materials within a qrtPCR reaction tube would mask fluorescent signals and therefore should be excluded. Counter to this, a few groups have now reported qrtPCR assays wherein filter paper is present in the reaction tube during qrtPCR. Significantly each of these groups has reported that the presence of filter paper increases background fluorescence.
Thus, prior to the instant disclosure a need in the art existed for a simplified qrtPCR assay, wherein the background fluorescent observed when filter paper is present is mitigated. The instant disclosure solves this problem and more.